The precursor to corticotropin, beta-endorphin and alpha-melanocyte stimulating hormone is found in both anterior and intermediate lobes of the pituitary and in the hypothalamus but it is processed to different hormones in each of these tissues. We have been trying to determine if the precursor has the same structure in each of these tissues. Poly-AMP enriched RNA as been isolated from each tissue and partially purifed by affinity chromatography using a cloned cDNA fragment (complementary to precursor mRNA) as the ligand. The partially purified precursor mRNA will be transcribed into cDNA with reverse transcription and cloned. The cloned cDNA will be sequenced to derive the amino sequence of the precursor in each of the three tissues. The synthesis and release of ACTH and beta-endorphin in pituitary tumor cells and in rat and mouse anterior pituitary cultures are stimulated by partially purified corticotropin releasing factor and inhibited by glucocorticoids. We are trying to determine the site of action of these regulators in culture systems by studying their effects on (1) the rate of transcription of precursor genes in isolated nuclei, (2) the nuclear processing of the precursor mRNA and the level of cytoplasmic mRNA and (3)the rate of translation of the mRNA. (The mRNA will be detectable by hybridization with cloned cDNA probes). We are also attempting to determine the site of action of the dopamine in inhibiting release of alpha-melanocyte stimulating hormone and beta-endorphin in rat intermediate lobe cultures.